The emergence of drug-resistant HIV variants constitutes a significant clinical challenge that necessitates the continued development of new antiviral agents, including small molecule CCR5 and CXCR4 antagonists. Recently, we have conferred genetic resistance to HIV infection through the targeted, permanent disruption of the ccr5 gene in human T cells through the use of highly specific zinc-finger nucleases (ZFNs). In vitro, cells with disrupted ccr5 grow normally, are resistant to infection by R5 virus strains, but are sensitive to X4 virus strains. NOG mice engrafted with R5-ZFN treated CD4+ T cells and subsequently infected with an R5 virus strain exhibited lower virus loads, and cells with disrupted ccr5 had a significant survival advantage in vivo. Our hypotheses for Project 3 are: 1. ZFN modification of both CXCR4 and CCR5 in primary CD4 T cells will protect cells from HIV infection while limiting genetic drift toward X4 strains in the NOG model of adoptive transfer, 2. Reconstitution of NOG mice with CCRS-modified CD34+ stem cells will broaden resistance to CCR5-tropic HIV, and 3. ZFN modification of CCR5 in CD4 T cells for adoptive transfer is advantageous over small molecule inhibitors in the setting of resistance to inhibitor In this project, we will continue our productive collaboration with Sangamo Biosciences, and through an iterative process test a panel of CXCR4-specific ZFNs (X4-ZFN) for their ability to disrupt the gene encoding this significant HIV coreceptor. The X4-ZFNs that disrupt CXCR4 most efficiently and specifically (Aim #1), determined in part through working with the Core C, and the lead CCR5 ZFNs will be tested for the ability to confer protection of cell lines and primary human T cells to infection by a variety of HIV-1 strains in vitro (Aim #2), and in vivo using the NOG mouse system in collaboration with Core B (Aim #3). We will also combine our X4- and R5-ZFNs in an attempt to render cells completely resistant to HIV-1 infection. While we will target CCR5 in both T cells and hematopoietic stem cells, we will limit the targeting of CXCR4 to T cells since CXCR4 has other important functions in hematopoiesis (Aims #2 and #3). Because genetic ablation of viral coreceptors imposes a selective pressure on virus that is quite distinct from the use of small molecule coreceptor antagonists, we will monitor our in vitro and in vivo infection experiments for the evolution of viral resistance (Aim #4), and if observed characterize the mechanism(s). Our long-term goal is to administer coreceptor-deficient autologous T cells and stem cells to HIV infected individuals.